| Objective: Diabetic kidney disease(DKD) is an serious microvascular complications of diabetes. The pathogenesis of diabetic kidney disease is very complex. Oxidative stress(OS), inflammation, susceptible gene, renal hemodynamic changes and many other factors have been suggested to contribute to the development of DKD. Brown Lee reported the unifying hypothesis : the induction of oxidative stress by the hyperglycemia plays a significant role in diabetes and its complications(1). There is considerable evidence that NADPH oxidase which exists across the vascular endothelial cells, glomerular mesangial cells and podocyte, is the major enzyme to produce oxygen free radicals(ROS), and there is an association between G338 A polymorphism of the p47 phox gene and the production of the ROS(2). 8-Hydroxy-2- deoxyguanosine(8-OHd G), for its stable quantity of production and less influence factors, is generally considered as an ideal marker of DNA oxidative injury degree in body.(3). Therefore, the aims of this study are following. Firstly, the enzyme-linked immunosorbent assay(ELISA) was used in this study to measures the serum 8-OHd G content in order to acquaint the DNA oxidative injury degree in the type 2 diabetes mellitus(T2DM). Secondly,The polymerase chain reaction(PCR) and single nucleotide polymorphism(SNP) direct sequencing were used to observed the relationship between DKD and p47 phox G338A gene polymorphism, which would be helpful to provide the theoretical basis of molecular genetics for diagnosis, prevention and treatment of DKD.Methods: According the T2 DM diagnosis and classification standard proposed from the WHO in 1999, a total of unrelated 198 T2 DM patients were selected from the Endocrinology department in the third hospital of Hebei medical university. These patients were divided into three groups according to the urinary albumin/creatinine ratio(urinary A / Cr), group A: simple diabetes consisted of 43 males and 27 females, with age 56.20±7.74 years, course 6.02±2.08 years; group B: Micro albuminuria consisted of 41 males and 24 females, with age 58.09±9.42 years, course 9.13±4.59 years; group C: Macro albuminuria consisted of 39 males and 24 females, with age 57.89 ±11.17 years, course 10.47±5.21 years. 70 healthy individuals in control group were collected from the medical center of the third hospital of Hebei Medical University during the same period, including 42 males and 28 females, with age of 55.60±10.81 years. As Han people in Hebei, they were unrelated to each other. The constituent ratios of the age and gender between the normal control group and the test group were matched.After overnight fasting, a 3-ml cubital vein blood sample was collected for extracting genomic DNA. A 4-ml vein blood sample was taken for detecting the biochemical indicators and 8-OHd G.(1) The measurement of clinical indicators: gender, age, the duration of diabetes, blood pressure, height, weight and body mass index(BMI) were measured and calculated.(2) The blood biochemistry data: Fasting blood glucose(FBG), total cholesterol(TC), triglycerides(TG), high density lipoprotein cholesterol(HDL-C), and low density lipoprotein cholesterol(LDL-C) were measured by automatic biochemical analyzer.(3) The measurement of Hb A1 c and urinary A / Cr: Hb A1 c and urinary A / Cr were measuered by Immunonephelometry.(4) The measurement of 8-OHd G: Serum level of 8-OHd G was measuered by enzyme linked immunosorbent assay(ELISA).(5) Detection of G338 A polymorphism ①genomic DNA extraction: 3ml 2% EDTA was used for anticoagulation. Leukocytes DNA was extracted by blood genomic DNA miniprep kit, then the spectrophotometer determined the concentration and purity of DNA. ②Deploy polymerase chain reaction and DNA direct sequeneing to detect p47 phox gene G338 A loci polymorphism. The forward 5’-TCCCAAGTGGTTTGACGG-3’ and reverse 5’- CCTCCTCTTTCTGGCTGTG-3’, the amplication was performed in a 25 ul volume containing 12.5ul PCR Premix, 8.5ul sterilized distilled water, upstream and downstream primers 1ul, 2ul of DNA. Samples were initially denatured at 94 for ℃ 3 minutes, followed by 30 cycles of denaturing at 94 ℃for 30 seconds, annealing at 54℃30 seconds, extension at 72 for ℃ 40 seconds and at a final step extended at 72 ℃for 3 minutes. ③ Genomic DNA purification. ④Deploy DNA direct sequencing to detect the genotype, then the genotype of p47 phox G338A were judged according to the patterns of DNA bands: GG, GA, AA.(6) Statistical analysis: All datas were analyzed by software SPSS13.0 version. Chi-square analysis was used to test whether p47 phox genotype distribution followed the Hardy-Weinberg equilibrium; Measurement data with normal distribution were expressed as mean±standard deviation( X ±s); ANOVA was performed to compare measurement data between groups; Chi-square analysis was used to compare enumeration data between groups; The relevance analysis of 8-OHd G concentration and other factors was estimated by the Spearman correlation analysis; The contribution of risk factors for type 2 diabetic nephropathy was measured by multivariable stepwise regression statistical method.Results: ①There was no significant difference in constituent ratios of the age and gender between T2 DM group and NC group. Compared with that data in NC group, the levels of BMI, SBP, DBP, FBG, TC, TG and LDL-C were significantly higher in the T2 DM group,but HDL-C was significantly lower(P<0.05 or P<0.01); Compared with that data in SDM, the patients with diabetic kiney disease had higher levels of course, BMI, DBP, TC, TG and lower HDL-C level(P<0.05 or P< 0.01); Compared with the data in microalbuminuria group,the levels of course, SBP, DBP were significantly higher in the macroalbuminuria group, the level of LDL-C was lower, but the HDL-C level has no significant difference between DKD groups ②The serum level of 8-OHd G in SDM was 5.02±0.69ng/ml, which was higher than that in NC group(3.03±0.64ng/ml)(P<0.05); The patients with DKD had more higher serum level of 8-OHd G than that in SDM and NC group(P<0.01), and compared with microalbuminuria group(7.44±0.97ng/ml), the levels of 8-OHd G(9.33±1.71ng/ml)in the macroalbuminuria group was significantly higher(P<0.05). ③The serum level of 8-OHd G was significantly positively correlated with course(r=0.264, P=0.001), SBP(r=0.195, P=0.004), FBG(r=0.217, P=0.000), Hb A1c(r=0.198, P=0.005), TC(r=0.173, P=0.008), TG(r=0.296, P=0.000), LDL-C(r=0.187, P=0.007) and significantly negatively correlated with HDL-C(r=-0.271, P=0.000). ④There was no significant difference in the freguencies of allele A and genotype GA+AA between SDM(18.6%, 10.0%)and NC group(11.4%, 5.7%); The genotype frequencies of GA+AA and allele A in DKD groups(32.3%,17.7% and 38.1%,21.4%) was significantly higher than that in the SDM and the NC group(P<0.05 or P<0.01), but there was no significant difference in that freguency berween two DKD groups.⑤The level of Hb A1 c, FBG, SBP and 8-OHd G in GA+AA genotype group were higher than that in GG genotype group(P<0.05 or P<0.01). The mobidity of DN in the GG+AA group(68.2%) was higher than that in GG group(X2=14.63,P<0.01). The incidence of diabetic kidney disease in genotype GA+AA is 3.072 times than that in genotype AA(OR=3.072, 95%CI 1.705-5.537).⑥Multivariable stepwise regression analysis showed that the main risk factors of DKD were course(β=0.257,P=0.036), LDL-C(β=0.357,P=0.015), SBP(β=0.291,P=0.019), FBG(β=0.285,P=0.022), and 8-OHd G(β=0.473,P=0.008).Conclusions:1 The serum 8-OHd G in T2 DM patients was higher, oxidative stress exists in T2 DM patients.2 The p47 phox G338A polymorphism exists in Hebei Han population with T2 DM, the individuals with allele A had higher serum 8-OHd G3 The 8-OHd G was an independent risk factor for diabetic kidney disease, but the allele A may not be the susceptible gene of diabetic kidney disease. |
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